Topical application of AMPK activators for pharmacological prevention of chronic pain

ABSTRACT

A topical composition containing an adenosine monophosphate protein kinase (AMPK) activator and methods of administering the AMPK activator for treating acute pain or preventing the development of chronic pain in a subject. The AMPK activator is resveratrol. A second AMPK activator may also be used. The second AMPK activator is metformin. The topical composition may be administered as a gel, ointment, cream, lotion, suspension, liquid, or transdermal patch.

CROSS REFERENCE

This application claims priority to and is a non-provisional of U.S.Provisional Patent Application No. 61/840,886, filed Jun. 28, 2013, thespecification(s) of which is/are incorporated herein in their entiretyby reference.

GOVERNMENT SUPPORT

This invention was made with government support under grant numberR01GM102575 awarded by the National Institutes of Health/NIGMS. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to a topical composition for treatingpain. In particular, the present invention relates to a topicalcomposition comprising an adenosine monophosphate protein kinase (AMPK)activator and methods for using the same. The present invention featuresa topical cream containing resveratrol with the anti-diabetic agentmetformin for treatment at an incision site.

BACKGROUND OF THE INVENTION

Chronic pain following surgical incisions is a major clinical problem.As used herein, the term “chronic pain” refers to pain lasting at leastthree (3) months and typically at least six (6) months, e.g., aftersurgery, injury or a disease. Currently available therapeutics given foracute post-surgical pain (opiates and non-steroidal anti-inflammatorydrugs, i.e., NSAIDs) do not always prevent the transition to chronicpost-surgical pain. Furthermore, opiates and NSAIDs can be habit formingand/or patients develop tolerance to these drugs, thereby requiringincreased dosage. Moreover, because these drugs are typicallyadministered orally, many patients suffer adverse gastrointestinaldiscomfort.

Therefore, there is a need to develop a composition that can treat painwithout some of the disadvantages exhibited by opiates and NSAIDs. Theaim is to establish AMPK activation as a mechanism for the alleviationof post-surgical, and possibly other persistent pain states and toutilize novel therapeutics that employ this mechanism of action of usein humans. Dysregulated protein translation regulation pathways thatunderlie persistent pain states can be negatively regulated byactivation of endogenous signaling factor AMPK.

The present invention features a novel therapeutic based on activationof adenosine monophosphate protein kinase (AMPK) to treat both acutepost-surgical pain and prevent the transition to chronic pain. AMPKactivators that possess different mechanisms of AMPK activation todemonstrate a shared endpoint, such as metformin and resveratrol, areused to accomplish this. Co-treatment with metformin and resveratroldoses at the time of incision block acute hypersensitivity andhyperalgesic priming suggesting potential super-additive effects ofcombined AMPK activator use.

SUMMARY OF INVENTION

Some aspects of the invention provide a topical composition for treatingpain. In some embodiments, the topical composition of the invention isuseful in treating acute post-surgical pain and/or preventing thetransition to chronic pain. Unlike conventional topical pain treatmentcreams that have either an opiate or an NSAID, the topical compositionof the invention comprises an adenosine monophosphate kinase (AMPK)activator. In one particular embodiment, the topical composition of theinvention comprises resveratrol.

Yet other aspects of the invention include treating pain by topicalapplying the composition of the invention at the surgical incision site(or the site of pain due to injury, etc.) in combination with a secondAMPK activator, such as metformin. The second AMPK activator can beadministered systemically or it can also be applied topically. In fact,the second AMPK activator can be a part of the topical compositionitself. Advantages of this strategy include topical administration,which prevents the occurrence of side effects due to systemic delivery,and the use of a widely prescribed, safe, and well-tolerated drug (e.g.,metformin or other AMPK activators that are currently in use).

One particular aspect of the invention provides a topical formulationcapable of treating pain comprising a therapeutically effective amountof an adenosine monophosphate kinase (AMPK) activator. In someembodiments, said AMPK activator comprises resveratrol. Yet in otherembodiments, the topical composition of the invention includes a secondAMPK activator. In some instances, the second AMPK activator comprisesmetformin.

The topical formulation can be in the form of a cream, lotion, atransdermal patch or a liquid solution. However, it should beappreciated that the scope of the invention includes all currently knowntopical method of administering the composition as well as any othertopical administration methods that are developed.

Another aspect of the invention provides a method for treating acutepain, neuropathic pain, or preventing the development of chronic pain ina subject, said method comprising topically applying a therapeuticallyeffective amount of an adenosine monophosphate kinase (AMPK) activatorto a subject in need of such treatment. In some embodiments, the methodalso includes systemically administering a second AMPK activator to saidsubject. For example, one can administer the second AMPK activator viaorally, parenterally, subcutaneously, sublingually, etc. Still in otherinstances, the second AMPK activator can also be administered topicallyto said subject. In fact, in some instances the topical compositioncomprises a mixture of the first and the second AMPK activators suchthat a single application is used to apply both AMPK activatorssimultaneously.

Yet in other embodiments, said second AMPK activator comprisesmetformin.

Compositions and methods of the invention can be used to treat apost-surgical pain, neuropathic pain, or to prevent post-surgical pain(or any other pain, e.g., due to injury or disease) from developing intoa chronic post-surgical pain.

Still in other embodiments, the AMPK activator is administered in atopical lotion or a topical cream formulation, a transdermal patch or asa solution that is applied to the site of pain or surgical incision.

Yet another aspect of the invention provides an adenosine monophosphatekinase (AMPK) activator composition comprising a combination of:

-   -   a topical composition formulation comprising a first AMPK        activator; and    -   a non-topical composition formulation comprising a second AMPK        activator, wherein said second AMPK activator is different from        the first AMPK activator.

In one particular embodiment, the composition is a mixture of a topicalcomposition comprising resveratrol and a systemically deliverable secondAMPK activator. In some instances, the second AMPK activator comprisesmetformin.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent application contains at least one drawing executed in color.Copies of this patent or patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

FIG. 1 shows that topical application of resveratrol inhibits acutemechanical hypersensitivity and hyperalgesic priming induced by plantarincision. Animals received a plantar incision on the left hind paw.Resveratrol (100 μg) or vehicle was applied on the paw on and around theincision either once, immediately following incision (A, B) or twice,immediately following incision as well as 24 hrs following incision (C,D). Mice were maintained under anesthesia with isoflurane for 1 hr untilthe drug was absorbed. A) Topical resveratrol (100 μg) applicationimmediately following incision resulted in blunted incision-inducedacute hypersensitivity B) Topical application of resveratrol (100 μg)immediately following incision abolished plantar incision-inducedhyperalgesic priming precipitated by PGE₂ injection on day 14 afterincision. C) Topical resveratrol (100 μg) application immediatelyfollowing incision and at 1 day post incision significantly blockedplantar incision-induced acute hypersensitivity D) Topical applicationof resveratrol (100 μg) at the time of incision and at 1 day postincision significantly blocked plantar incision-induced hyperalgesicpriming precipitated by PGE₂ injection on day 14 after incision.

FIG. 2 shows metformin inhibits acute mechanical hypersensitivityinduced by plantar incision. Animals received a plantar incision on theleft hind paw. Metformin (30, 100, 150 or 200 mg/kg) or vehicle wasinjected intra-peritonially for 4 days starting 2 days prior to thesurgery. A) Metformin (30, 100, 150 or 200 mg/kg) injectionsignificantly blocked plantar incision-induced acute hypersensitivity.B) Area under the curve (AUC) analysis showing dose-related effects inA.

FIG. 3 shows metformin inhibits plantar incision-induced hyperalgesicprecipitated by PGE₂ injection. Animals received a plantar incision onthe left hind paw. Metformin (30, 100, 150 or 200 mg/kg) or vehicle wasinjected intra-peritonially for 4 days starting 2 days prior to thesurgery. Hyperalgesic priming was precipitated by a second intraplantarinjection of inflammatory mediator PGE₂ (100 ng) in the hind paw on day14 after incision. A) Metformin (30, 100, 150 or 200 mg/kg) injectionsignificantly blocked plantar incision-induced hyperalgesic primingprecipitated by PGE₂ injection on day 14 after incision. B) Area underthe curve (AUC) analysis showing dose-related effects in A.

FIG. 4 shows OSU-53 inhibits acute mechanical hypersensitivity inducedby plantar incision. Animals received a plantar incision on the lefthind paw. OSU53 (10 or 30 μg) or vehicle was injected locally into theleft hind paw around the incision immediately following incision and 24hrs post-surgery. A) OSU53 (10 or 30 μg) injection significantly blockedplantar incision-induced acute hypersensitivity. B) Area under the curve(AUC) analysis showing dose-related effects in A.

FIG. 5 shows OSU-53 inhibits plantar incision-induced hyperalgesicprecipitated by PGE₂ injection. Animals received a plantar incision onthe left hind paw. OSU53 (10 or 30 μg) or vehicle was injected locallyinto the left hind paw around the incision immediately followingincision and 24 hrs post-surgery. Hyperalgesic priming was precipitatedby a second intraplantar injection of inflammatory mediator PGE₂ (100ng) in the hind paw on day 14 after incision. A) OSU53 (10 or 30 μg)injection significantly blocked plantar incision-induced hyperalgesicpriming precipitated by PGE₂ injection on day 14 after incision. B) Areaunder the curve (AUC) analysis showing dose-related effects in A.

FIG. 6 shows A-769662 does not significantly inhibit acute mechanicalhypersensitivity induced by plantar incision but blocks the hyperalgesicprecipitated by PGE₂ injection. Animals received a plantar incision onthe left hind paw. A-769662 (30 mg/kg) or vehicle was injectedintra-peritonially for 4 days starting 2 days prior to the surgery. A)A-769662 (30 mg/kg) injection did not block plantar incision-inducedacute hypersensitivity. B) A-769662 (30 mg/kg) injection significantlyblocked plantar incision-induced hyperalgesic priming precipitated byPGE₂ injection on day 14 after incision.

FIG. 7 shows co-treatment with systemic metformin and local resveratrolinhibits acute mechanical hypersensitivity induced by plantar incision.Animals received a plantar incision on the left hind paw. Metformin (150μg) was injected intra-peritonially for 4 days starting 2 days prior tothe surgery and resveratrol (3 μg) was injected in the left hind pawaround the incision on the day of the surgery and 24 hrs post-surgery.A) Co-treatment with systemic metformin and local resveratrolsignificantly blocked plantar incision-induced acute hypersensitivity.B) Area under the curve (AUC) analysis showing dose-related effects in Acompared to systemic metformin (150 μg) or local resveratrol (3 μg)alone.

FIG. 8 shows co-treatment with systemic metformin and local resveratrolinhibits plantar incision-induced hyperalgesic precipitated by PGE₂injection. Animals received a plantar incision on the left hind paw.Metformin (150 μg) was injected intra-peritonially for 4 days starting 2days prior to the surgery and resveratrol (3 μg) was injected in theleft hind paw around the incision on the day of the surgery and 24 hrspost-surgery. A) Co-treatment with systemic metformin and localresveratrol significantly blocked plantar incision-induced hyperalgesicpriming precipitated by PGE₂ injection on day 14 after incision. B) Areaunder the curve (AUC) analysis showing dose-related effects in Acompared to systemic metformin (150 μg) or local resveratrol (3 μg)alone.

FIG. 9 shows AMPK activators don't appear to affect wound healingnegatively in a model of post-surgical pain. Animals received a plantarincision on the left hind paw. Skin from the left hind paw was excised 3and 7 days after plantar incision and wound healing and wound closurewas assessed using Hematoxylin & Eosin (H & E) staining. A) Resveratrol(10 μg) or vehicle was injected in the left hind paw around the incisionon the day of the surgery and 24 hrs post-surgery. There was nodifference in the wound size between the resveratrol and vehicle treatedgroups on Day 3. The wound was completely closed by Day 7 in the animalsin both groups receiving vehicle or resveratrol. B) Metformin (200mg/kg) or vehicle was injected intra-peritonially for 4 days starting 2days prior to the surgery. No differences were noted in the wound sizebetween the metformin and vehicle treated groups on Day 3. The wound wascompletely closed by Day 7 in the animals in both groups receivingvehicle or metformin. C) OSU53 (10 or 30 μg) or vehicle was injectedlocally into the left hind paw around the incision immediately followingincision and 24 hrs post-surgery. There was no difference in the woundsize between the OSU53 and vehicle treated groups on Day 3. The woundwas completely closed by Day 7 in the animals in both groups receivingvehicle or OSU53.

DETAILED DESCRIPTION OF THE INVENTION

Surgery is a major cause of persistent pain suggesting that treatmentsthat directly target the molecular pathology promoting post-surgicalpain, particularly those that contribute to the progression to chronicpain, are needed. The present inventors have previously demonstratedthat dysregulated protein translation regulation pathways, in particularERK/eIF4E and mTOR signaling pathways underlie persistent pain statesand that AMPK activators can profoundly inhibit ERK and mTOR signalingin sensory neurons. Furthermore, the present inventors have discoveredthat local injection of resveratrol, a potent AMPK activator, into thehindpaw following plantar incision dose-relatedly reversesincision-mediated mechanical hypersensitivity as well as hyperalgesicpriming induced by incision.

Surprisingly and unexpectedly, the present inventors have furtherdiscovered that a topical application of an AMPK activator, such asresveratrol, resulted in a bona-fide mechanism for the alleviation ofpost-surgical, and other persistent or chronic pain states.

Thus, some aspects of the present invention provide a topicalcomposition comprising an AMPK activator and using the same to treatpain associated with post-surgery as well as other acute and neuropathypain, including pain due to injury or disease. In addition, compositionsand methods of the invention can be used to prevent any acute injuryfrom developing into a chronic pain. In some embodiments of theinvention, the composition comprises a plurality of AMPK activators.Within these embodiments in some instances, the topical compositioncomprises resveratrol. In other embodiments other AMPK activator(s) suchas metformin, OSU-53, A-769662, other AMPK activators that are known toone skilled in the art, or a combination thereof can be included in thetopical composition. AMPK activators are well known to one skilled inthe art. See, for example, Merck Index, 15^(th) Ed., Edited by MaryadeleJ O'Neil, Royal Society of Chemistry, 2013, and Physicians' DeskReference (i.e., “PDR”) 67^(th)/Ed., 2013, all of which are incorporatedherein by reference in their entirety. Alternatively, other AMPKactivator can be administered systemically in combination with a topicalapplication of at least one of the AMPK activators, such as resveratrol.It should be noted, that AMPK activators that are used in combinationcan possess different mechanisms of AMPK activation.

The present invention features a topical formulation capable of treatingpain and preventing a transition into chronic pain. In some embodiments,the topical formulation comprises a therapeutically effective amount ofan adenosine monophosphate kinase (AMPK) activator. In some embodiments,the topical formulation comprises a second AMPK activator. The presentinvention also features a method for treating acute pain or preventingthe development of chronic pain in a subject, said method comprisingtopically applying a therapeutically effective amount of the AMPKactivator to a subject in need of such treatment. In some embodiments,the method further comprises a method of administering a second AMPKactivator to said subject. In some embodiments, the method ofadministering said second AMPK to said subject is selected from among:

-   -   a. administering topically;    -   b. administering systemically; or    -   c. administering parenterally.

In some embodiments, the AMPK activator comprises resveratrol. In someembodiments, the second AMPK activator comprises metformin. In someembodiments, the topical formulation further comprises a topical base,wherein the topical base is polyethylene glycol (PEG 400). In someembodiments, the topical formulation is in a form of a cream, a lotion,an ointment, a gel, a transdermal patch or a liquid solution.

In some embodiments, the pain comprises a post-surgical pain,neuropathic pain, or any pain potentially leading to the development ofchronic post-surgical pain.

In some embodiments, a concentration of resveratrol in the topicalformulation is between about 0.5 mg/ml and 5 mg/ml. In some embodiments,the concentration of resveratrol in the topical formulation is betweenabout 1 mg/ml and 3 mg/ml. In some embodiments, the concentration ofresveratrol in the topical formulation is between about 1.5 mg/ml and2.5 mg/ml.

In some embodiments, a concentration of metformin in the topicalformulation is between about 0.5 mg/ml and 5 mg/ml. In some embodiments,the concentration of metformin in the topical formulation is betweenabout 1 mg/ml and 3 mg/ml. In some embodiments, the concentration ofmetformin in the topical formulation is between about 1.5 mg/ml and 2.5mg/ml.

In some embodiments, the topical formulation is applied on and around anincision immediately following incision. In some embodiments, thetopical formulation is applied on and around an incision immediatelyfollowing incision and at about 24 hours following incision. In someembodiments, the topical formulation is applied on and around anincision between about 30 seconds and one minute following incision. Insome embodiments, the topical formulation is applied on and around anincision between about one minute and five minutes following incision.In some embodiments, the topical formulation is applied on and around anincision between about five minutes and ten minutes following incision.In some embodiments, the topical formulation is applied on and around anincision between about ten minutes and thirty minutes followingincision. In some embodiments, the topical formulation is applied on andaround an incision between about thirty minutes and one hour followingincision. In some embodiments, the topical formulation is applied on andaround an incision about every three to four hours following incision.In some embodiments, the topical formulation is applied on and around anincision about 24 hours following incision. In some embodiments, thetopical formulation is applied on and around an incision about 24 hoursfollowing incision.

In some embodiments, the topical formulation is applied to an incisionsite at about 24 hours prior to incision. In some embodiments, thetopical formulation is applied to an incision site at about 48 hoursprior to incision. In some embodiments, the topical formulation isapplied to an incision site at about every four to six hours for abouttwo days prior to incision. In some embodiments, the topical formulationis applied on and around an incision site at about every two to fourdays, starting at about two days prior to incision, and up to thefourteenth day following incision.

In some embodiments, the dosage of resveratrol is between about 1 mg/kgto about 5 mg/kg. In some embodiments, the dosage of resveratrol isbetween about 5 mg/kg to about 10 mg/kg. In some embodiments, the dosageof resveratrol is between about 10 mg/kg to about 15 mg/kg. In someembodiments, the dosage of resveratrol is between about 15 mg/kg toabout 25 mg/kg. In some embodiments, the dosage of resveratrol isbetween about 25 mg/kg to about 35 mg/kg. In some embodiments, thedosage of resveratrol is between about 35 mg/kg to about 50 mg/kg. Insome embodiments, the dosage of resveratrol is between about 35 mg/kg toabout 50 mg/kg.

In some embodiments, the dosage of metformin is between about 1 mg/kg toabout 5 mg/kg. In some embodiments, the dosage of metformin is betweenabout 5 mg/kg to about 10 mg/kg. In some embodiments, the dosage ofmetformin is between about 10 mg/kg to about 20 mg/kg. In someembodiments, the dosage of metformin is between about 20 mg/kg to about30 mg/kg. In some embodiments, the dosage of metformin is between about30 mg/kg to about 50 mg/kg. In some embodiments, the dosage of metforminis between about 50 mg/kg to about 100 mg/kg. In some embodiments, thedosage of metformin is between about 100 mg/kg to about 150 mg/kg. Insome embodiments, the dosage of metformin is between about 150 mg/kg toabout 200 mg/kg. In some embodiments, the dosage of metformin is betweenabout 200 mg/kg to about 250 mg/kg. In some embodiments, the dosage ofmetformin is between about 250 mg/kg to about 300 mg/kg.

In one particular embodiment, a topical composition comprisingresveratrol is prepared as a cream. Briefly, lyophilized resveratrol wasserially dissolved in polyethylene glycol 400 (PEG 400) and solid PEGointment to achieve a final concentration of 2 mg/ml. Using the plantarincision model in mice, the present inventors demonstrated that topicalapplication of resveratrol attenuated incision-induced mechanicalhypersensitivity as well as the development of hyperalgesic primingprecipitated by hind paw injection of PG_(E2) following resolution ofincision-induced mechanical hypersensitivity. Metformin, which isclinically available and widely prescribed, stimulated upstream LKB1activity to activate AMPK whereas OSU-53 and A-769662 are positiveallosteric modulators that directly activate AMPK.

Using the Brennan incision model in mice, the present inventors havedemonstrated that systemic metformin or local OSU-53 injectiondose-dependently and efficaciously attenuated incision-inducedmechanical hypersensitivity as well as the development of hyperalgesicpriming precipitated by hind paw injection of PGE₂ following resolutionof incision-induced mechanical hypersensitivity. Interestingly, systemicA-769662 was not effective in blocking incision-induced acute mechanicalhypersensitivity; however it significantly blocked hyperalgesic priming.This effect was paralleled by lower doses of metformin, which had noacute effect yet blocked hyperalgesic priming. Finally, co-treatmentwith systemic metformin and local resveratrol at individuallysub-efficacious doses at the time of incision blocked acutehypersensitivity and hyperalgesic priming suggesting potentialsynergistic or super-additive effects of combined AMPK activator use.None of these treatment approaches adversely affected wound healing.These results provide further evidence for activation of AMPK as a noveltreatment avenue for acute and chronic pain states induced by surgery.

Post-surgical pain has been identified as a potential major cause forchronic pain. Between 10 and 50% of patients who undergo surgery developchronic pain following surgical procedures such as groin hernia repair,breast and thoracic surgery, leg amputation, or coronary artery bypasssurgery (Kehlet et al., 2006) and up to a quarter of all chronic painpatients suffer from persistent pain because of a prior surgery (Crombieet al., 1998). This chronic pain can be debilitating in 2-10% of thispopulation (Johansen et al., 2012; Kehlet et al., 2006). Despite thefact that analgesics for the treatment of acute post-surgical pain arewidely available, surgery remains a major cause of persistent painsuggesting that treatments that directly target the molecular pathologyof post-surgical pain, particularly those that prevent the transition tochronic post-surgical pain, are needed. Recent advances in understandingof the mechanisms of post-surgical pain have led to the elucidation ofsignaling pathways and mediators that play an important role in drivingpost-surgical pain. However, treatment approaches that target thesepathways or mediators are not currently clinically utilized.

The present inventors have previously demonstrated that dysregulatedprotein translation regulation pathways, in particular ERK and mTORsignaling pathways, underlie persistent pain states such as neuropathicpain. There is an upregulation of the mTOR and ERK signaling pathways inneuropathic pain models in rodents and ectopic activity of theperipheral nerves (Geranton et al., 2009; Huang et al., 2008;Jimenez-Diaz et al., 2008). In model of post-surgical pain, there is anincrease in IL-6 and NGF levels in the serum and skin around theincision (Banik et al., 2005; Bryan et al., 2005; Matsuda et al., 1998;Sato and Ohshima, 2000). IL-6 and NGF, in turn, signal through ERK andmTOR pathways inducing nociceptive sensitization (Melemedjian et al.,2010). The present inventors have recently demonstrated that both thesepathways can be negatively regulated by an endogenous signaling factor,adenosine monophosphate protein kinase (AMPK). AMPK is a ubiquitousenergy-sensing kinase which can be activated physiologically by increasein intracellular AMP/ATP ratio which occurs during energy deprivation orcell starvation. AMPK can be activated pharmacologically as well by anumber of clinically available drugs, e.g., metformin or naturalproducts such as resveratrol. AMPK can also be activated by a number ofinvestigational compounds e.g. AICAR, A769662 or OSU53. The presentinventors have demonstrated that the AMP activated protein kinase (AMPK)activators, metformin and A769662, inhibited translation regulationsignaling pathways and nascent protein synthesis in injured nervesneurons resulting in a resolution of neuropathic allodynia induced byperipheral nerve injury. In addition, the present inventors alsodemonstrated that resveratrol, a potent and efficacious activator ofAMPK, profoundly inhibits ERK and mTOR signaling in sensory neurons in atime- and concentration-dependent fashion and local injection ofresveratrol around the surgery site attenuates the surgery induced acutemechanical hypersensitivity and persistent hyperalgesic priming in amodel of post-surgical pain.

The present inventors have discovered that AMPK activation is abona-fide mechanism for the alleviation of post-surgical, and otherpersistent pain states. Some aspects of the invention are based on thisdiscovery to provide novel therapeutics and therapeutic strategies thatemploy this mechanism of action for use in mammals such as humans. Asdiscussed in the Examples section, the present inventors have utilizedmultiple AMPK activators, including resveratrol, metformin, OSU-53 andA-769662, which possess different mechanisms of AMPK activation todemonstrate a shared endpoint—inhibition of incision-induced mechanicalhypersensitivity and hyperalgesic priming.

The advantages of the topical route of drug administration include:avoidance of the risks and inconvenience of parenteral treatment;avoidance of the variable absorption and metabolism associated with oraltreatment; continuity of drug administration, permitting use ofpharmacologically active agents with short biological half-lives;potential reduction of gastrointestinal irritation in systemicadministration; and treatment of cutaneous manifestations of diseasesusually treated systemically.

In some embodiments, an AMPK activator is formulated for topicaladministration to the epidermis as a solution, gel, ointment, cream,suspension, lotions, a transdermal patch etc. as are well-known in theart. Ointments and creams can, for example, be formulated with anaqueous or oily base with the addition of suitable thickening and/orgelling agents. Lotions can be formulated with an aqueous or oily baseand will in general also contain one or more emulsifying agents,stabilizing agents, dispersing agents, suspending agents, thickeningagents, or coloring agents. Formulations suitable for topicaladministration in the mouth include lozenges comprising active agents ina flavored base, usually sucrose and acacia or tragacanth; pastillescomprising the active ingredient in an inert base such as gelatine andglycerine or sucrose and acacia; and mouthwashes comprising the activeingredient in a suitable liquid carrier.

In some embodiments, the topical formulation is a liquid, for example, ahomogeneous liquid or a suspension, sold in a bottle which dispenses theformulation as drops or a liquid film (for example, from an applicatortip that contacts a target area of the skin to dispense the liquidsubstantially only on a target area of the skin to be treated). In oneembodiment, the formulation is a cream or ointment, sold in a tube whichdispenses the formulation to a target area of the skin. In anotherembodiment, the AMPK activator is provided in a viscous liquid (such ascarboxylmethylcellulose, hydroxypropyl-methycellulose, polyethyleneglycol, glycerin, polyvinyl alcohol, or oil containing drops) forrubbing into the skin. The formulations may have preservatives or bepreservative-free (for example in a single-use container). Oneembodiment is any of the aforementioned formulations in a kit fortopical or local administration.

Particular embodiments of the topical formulations include atherapeutically effective amount of the AMPK activator, a topical base,an antioxidant, an emollient, and an emulsifier. The topical base maycomprise polyethylene glycol having a selected molecular weight.Particular embodiments comprise a polyethylene glycol having a molecularweight of from 3000 to 8000 daltons as a topical base. Topical basesinclude, but are not limited to, hydrophobic vehicles such ashydrocarbons, liquid petrolatum (mineral oil, liquid paraffin, paraffinoil), white petrolatum (petroleum jelly, VASELINE®), yellow petrolatum(petroleum jelly), squalane (perhydrosqualene, spinacane), andsilicones; silicones such as liquid polydimethylsiloxanes (dimethicone,silastic, medical grade silicone oil), alcohols such as lauryl alcohols(1-dodecanol, dodecyl alcohols), myristyl alcohols, (tetradecanol,tetradecyl alcohols), cetyl alcohols (hexadecanol, ethal, palmitylalcohols), stearyl alcohols (stenol, cetosteryl alcohols), oleylalcohols (ocenol); sterols such as sterol esters; lanolin such ashydrous wool fat, lanum; anhydrous lanolin (such as wool fat, anhydrouslanum, agnin); semi synthetic lanolins; carboxylic acids such as lauricacid, myristic acid, palmitic acid, stearic acid, oleic acid; esters andpolyesters, such as cholesterol esters (stearate), ethylene glycolmonoesters, propylene glycol monoesters, glyceryl monoesters, glycerylmonostearate, sorbitol monoesters, sorbitan monoesters, sorbitoldiesters, sorbitan polyesters (spans, arlacels), glyceryl tristearate,lard, almond oil, corn oil, castor oil, cottonseed oil, olive oil,soybean oil, hydrogenated oils, sulfated oils, isopropyl myristate,isopropyl palmitate; and ethers and polyethers (polydisperse ormonodisperse), such as polyethylene glycols or polypropylene glycols(pluronics).

In certain embodiments, the formulation is an ointment, which is asemisolid preparation intended for external application to the skin ormucous membranes. In a specific example, the ointment is based onpetrolatum. The ointment does not contain sufficient water to separateinto a second phase at room temperature. A water-soluble ointment may beformulated with a water-miscible solvent. Ointments are ideal emollientswith good skin penetration and adherence to surfaces. The ointment is ina convenient container such as a tube or jars.

In some embodiments, the water-miscible solvent is polyalkylene glycolhaving an average molecular weight of from 200 daltons to 600 daltons.In certain embodiments the water-miscible solvent comprises PEG-400, andeven more particularly PEG-400 substantially free of impurities. Incertain embodiments, the PEG-400 comprises less than 65 ppmformaldehyde, less than 10 ppm formaldehyde, or 1 ppm or lessformaldehyde. In one aspect, the water-miscible solvent includesglycofurol, which can be used in place of or in combination with a lowmolecular weight polyalkylene glycol, particularly a polyethyleneglycol, such as PEG-400. Water-miscible solvents that may be used, butare not limited to, include polyols and polyglycols such as propyleneglycol (1,2-propanediol), glycerin (glycerol), liquid polyethyleneglycol, solid polyethylene glycol (hard macrogol, Carbowax®), glycolfurol, 1,2-phenol-hexanetriol, sorbitol solution, esters and polyesterssuch as polyoxyethylene sorbitan monoesters (e.g., Tween® 60) andpolyoxy ethylene sorbitan polyesters (e.g., Tween® 20), ethers andpolyethers such as polyethylene glycol monocetyl ether (cetomacrogol1000) and polyethylene-polypropylene glycols (pluronics). In oneembodiment, the water-miscible solvent includes PEG-400.

In some embodiments, the topical formulations for use as describedherein also can include a penetration enhancer to improve delivery ofthe AMPK activator into the skin. Suitable penetration enhancersinclude, but are not limited to, alcohol, alkyl methyl sulfoxide,pyrrolidone, laurocapram, dimethyl formamide, tetrahydrofurfurylalcohol, an amphiphile, or other miscellaneous enhancers such asclofibric acid amide, hexamethylene lauramide, dimethyl isosorbide,propylene glycol, proteolytic enzymes, terpenes or sesquiterpenes.

In some embodiments, the topical formulations for use as describedherein also can include a surfactant. Suitable surfactants include, butare not limited to a sterol or sterol ester, for example cholesterol(cholesterin), lanolin (hydrous wool fat, lanum), anhydrous lanolin(wool fat, anhydrous lanum, agnin), or semi synthetic lanolin;carboxylic acids such as Na+, K+, ethanolamine salts of lauric acid,myristic acid, palmitic acid, stearic acid, oleic acid, or an ether orpolyether such as polyethylene-polypropylene glycols (pluronics). If anoil-in-water (o/w) emulsifier is desired, the following examples may beused: esters and polyesters such as polyoxyethylene, sorbitan monoesters(Span™ 20, Span™ 40, Span™ 80), polyoxy ethylene esters(stearate-polyethylene glycol monoesters, Myrj® 45, Myrj® 59), polyoxyethylene sorbitan polyesters (tweens); ethers and polyethers such aspolyethylene glycol monocetyl ether (cetomacrogol 1000) orpolyethylene-polypropylene glycols (pluronics), and others such assodium lauryl sulfate, borax (sodium borate), ethanolamine, ortriethanolamine. Nonionic surfactants, like Surfactant 190 (dimethiconecopolyol), Polysorbate 20 (Tween® 20), Polysorbate 40 (Tween® 40),Polysorbate 60 (Tween® 60), Polysorbate 80 (Tween® 80), lauramide DEA,cocamide DEA, and cocamide MEA, amphoteric surfactants like Oleylbetaine and cocamidopropyl betaine (Velvetex® BK-35), and cationicsurfactants, like Phospholipid PTC (Cocamidopropyl phosphatidylPG-dimonium chloride), can be used. Appropriate combinations or mixturesof such surfactants may also be used.

In some embodiments, the topical formulation comprises an antioxidant.Examples of anti-oxidants include, but are not limited to, butylatedhydroxyanisole, butylated hydroxytoluene, ascorbic acid, a tocopherol,and combinations thereof, with particular embodiments comprisingbutylated hydroxytoluene as an antioxidant. In some embodiments, thetopical formulations can include at least one emollient. Examples ofsuitable emollients include, but are not limited to, volatile andnon-volatile silicone oils, highly branched hydrocarbons, and mixturesthereof.

In some embodiments, the topical formulation may further may includedyes/colorants and/or fragrances. Suitable fragrances and colors, suchas 0.05% to 0.25% (w/w) caramel colorant caramel, FD&C Red No. 40 andFD&C Yellow No. 5, may be used in the formulations. Other examples offragrances and colors suitable for use in topical products are known inthe art.

The topical formulation comprising the AMPK activator optionally maycomprise additional pharmaceutically acceptable ingredients such asadditional solvents, gelling agents, fragrances, preservatives,anti-bacterial agents, diluents, stabilizers and/or adjuvants. However,such optional materials must not unduly interfere with the transdermaldelivery of the drug active.

Examples of solvents include, but are not limited to, short chainalcohols and ethers; preferred optional solvent materials includepolyethylene glycols, dipropylene glycol, ethylene glycol monoethylether, ethanol, isopropanol, and dimethyl isosorbide, or water may alsobe used as a solvent or co-solvent in the compositions of thisinvention.

Suitable fragrances and colors, such as caramel, FD&C Red No. 40 andFD&C Yellow No. 5, may be used in the formulations. Other examples offragrances and colors suitable for use in topical products are known inthe art.

In some embodiments, the topical formulation may also include othersuitable additional and adjunct ingredients. Examples of suchingredients include, but are not limited to, absorbents (e.g.,hydrogels), astringents (e.g., witch hazel, alcohol, and herbal extractssuch as chamomile extract), binders (e.g., starch, cellulose ethers,microcrystalline cellulose, calcium hydrogen phosphate, calciumphosphate dibasic, and calcium sulfate dihydrate), other excipients(e.g., polyvinylpyrrolidone (PVP), tocopheryl polyethylene glycol 1000succinate (also known as vitamin E TPGS, or TPGS), dipalmitoylphosphatidyl choline (DPPC), trehalose, sodium bicarbonate, glycine,sodium citrate, and lactose), buffering agents (e.g., monobasic ordibasic potassium phosphate, monobasic or dibasic sodium phosphate,magnesium hydroxide), chelating agents (e.g., EDTA(ethylenediaminetetraacetic acid, tetrasodium salt)), film-formingagents (e.g., chitosan, hydroxypropylmethylcellulose, polyvinylalcohol), conditioning agents (e.g., petrolatum, glycerin, propyleneglycol), opacifying agents (e.g., titanium dioxide), pH adjusters (e.g.,citric acid and sodium hydroxide), and protectants.

While particular embodiments of the present invention have beendescribed, it will be obvious to those skilled in the art that variouschanges and modifications to the compositions disclosed herein can bemade without departing from the spirit and the scope of the invention.It is intended to cover, in the appended claims, all such modificationsthat are within the scope of this invention.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting. Inthe Examples, procedures that are constructively reduced to practice aredescribed in the present tense, and procedures that have been carriedout in the laboratory are set forth in the past tense.

EXAMPLES Materials and Methods

Experimental Animals:

Male ICR mice (Harlan, 20-25 g) were used for the study. All animalprocedures were approved by the Institutional Animal Care and UseCommittee of The University of Arizona and were in accordance withInternational Association for the Study of Pain guidelines.

Behavior Testing:

For the testing, animals were placed in acrylic boxes with wire meshfloors and allowed to habituate for approximately 1 h on all testingdays. Paw withdrawal thresholds were measured using calibrated von Freyfilaments (Stoelting, Wood Dale, Ill.) by stimulating the plantar aspectof left hind paw using the up-down method (Chaplan et al., 1994).

Plantar Incision and Behavioral Testing:

Prior to surgery all animals were assessed for paw withdrawalthresholds. A mouse model of incisional pain was used for this study(Banik et al., 2006). A 5 mm longitudinal incision was made with anumber 11 blade through skin, fascia and muscle of the plantar aspect ofthe hind paw in isoflurane-anesthetized rats. Sham controls underwentthe same procedure but without the incision. The skin was apposed with 2sutures of 5 mm silk. Animals received intraplantar injection ofresveratrol or vehicle around the incision at times indicated afterincision. Animals were allowed to recover for 24 hrs and then pawwithdrawal thresholds were measured at 24 hrs, 48 hrs, 5, 9, and 13 dayspost-surgery. For persistent sensitization experiments, the animalsreceived an intraplantar injection of PGE2 (100 ng/25 μl) 14 daysfollowing incision or sham procedures. The paw withdrawal thresholdswere again measured at 1 h, 3 h and 24 h following the PGE2 injection.

Immunohistochemistry:

To determine re-epithelialization, plantar skin was excised from theleft hind paw of mice either 3 days or 7 days following plantar incisionsurgery. The skin was immediately cryoprotected and frozen in O.C.Tcompound and sectioned (20 μm) on cryostat. The sections were then fixedin formalin and immersed in 0.1% hematoxylin for 3 minutes, washed intap water, then immersed in 0.1% eosin for 3 minutes, and dehydratedthrough graded ethanol (Protocol from University of Michigan Center forOrganogenesis). Finally sections were coverslipped with Permount(Fisher, Pittsburgh, Pa.). The sections were imaged and the widthbetween 2 epithelial edges was measured using Olympus BX51 microscope.

Drugs and Primary Antibodies:

Resveratrol was from Cayman Chemical; metformin from LKT laboratories.For the cream preparation with resveratrol, lyophilized resveratrol wasserially dissolved in polyethylene glycol 400 (PEG 400) and solid PEGointment to achieve a final concentration of 2 mg/ml. Hematoxylin Gill2X, O.C.T. compound, Permount mounting medium and xylene were purchasedfrom Fischer Scientific. Compound OSU 53 was synthesized by semi-manualsolid-phase peptide synthesis (Boitano et al., 2011; Flynn et al., 2011;Josan et al., 2008; Lee et al., 2011; Vagner et al., 2008) performed infritted syringes using a Domino manual synthesizer obtained from Torviq(Niles, Mich.). Crude peptides were purified by HPLC and size exclusionchromatography. Purity of the peptides was ensured using analytical HPLC(Waters Alliance 2695 separation model with a dual wavelength detector,Waters 2487) with a reverse-phase column (Waters Symmetry, 4.6×75 mm,3.5 μm; flow rate, 0.3 ml/min). Structures were characterized byelectrospray ionization on a Thermoelectron (Finnigan) LCQ ion trapinstrument (low resolution), a Bruker Ultraflex III MALDI-TOF/TOF (lowresolution), or a Bruker 9.4 T Fourier transform ion-cyclotron resonance(high resolution accurate mass) mass spectrometer.

Statistical Analysis and Data Presentation:

Data are shown as means and the standard error of the mean (±SEM) of 6animals (for behavioral studies). Graph plotting and statisticalanalysis used Graphpad Prism Version 5.03 (Graph Pad Software, Inc. SanDiego, Calif., USA). Statistical evaluation was performed by one- ortwo-way analysis of variance (ANOVA), followed by appropriate post-hoctests. The a priori level of significance at 95% confidence level wasconsidered at p<0.05.

RESULTS

Topical Resveratrol Inhibits Acute Mechanical Hypersensitivity andHyperalgesic Priming Induced by Plantar Incision:

The present inventors have previously demonstrated that local injectionof resveratrol, a potent AMPK activator, into the hind paw followingplantar incision dose-relatedly reverses incision-mediated mechanicalhypersensitivity as well as hyperalgesic priming induced by incision.Though resveratrol injection efficaciously blocked incision-inducedallodynia, for clinical translability there is a need of better route ofadministration which is convenient and causes minimal discomfort. Toaddress this issue, the present inventors made a novel cream preparationwith resveratrol which can be applied topically. Lyophilized resveratrolwas serially dissolved in polyethylene glycol 400 (PEG 400) and solidPEG ointment to achieve a final concentration of 2 mg/ml. A mouse modelwas utilized for incisional pain to assess if topical application ofresveratrol can prevent development of allodynia following the plantarincision (Brennan et al., 1996; Pogatzki and Raja, 2003). Animalsreceived a plantar incision on the left hindpaw. Resveratrol (100 μg) orvehicle was applied on the paw on and around the incision eitherimmediately following incision or immediately following incision and 24hrs following incision. Mice were maintained under anesthesia withisoflurane for 1 hr until the drug was absorbed. Mice with plantarincision that received vehicle displayed mechanical hypersensitivitylasting for at least 9 days. In contrast, animals that received topicalresveratrol at the time of incision displayed blunted hypersensitivitythough not significant (FIG. 1A). However, applying resveratroltopically twice i.e. at the time of incision and 24 hrs followingincision, significantly attenuated mechanical hypersensitivity inducedby incision (FIG. 1C). In this model, hyperalgesic priming can berevealed by a second intraplantar injection of inflammatory mediatorPGE₂ (100 ng) in the hind-paw, after the resolution of initial allodynia(Asiedu et al., 2011) following plantar incision. Topical application ofresveratrol at the time of incision or at the time of incision and 1 daylater both prevented the development of hyperalgesic primingprecipitated by hind paw injection of PGE₂ following resolution ofincision-induced mechanical hypersensitivity (FIGS. 1B and 1D). Thus,resveratrol not only blocks acute allodynia induced by plantar incisionbut it also blocks its transition to persistent nociceptive state. Theseresults suggest that even local application of resveratrol can be apotentially efficacious treatment for post-surgical pain.

Metformin Inhibits Acute Mechanical Hypersensitivity and HyperalgesicPriming Induced by Plantar Incision:

Another therapeutic opportunity for activating AMPK is the prototypicalAMPK activator, metformin. Metformin is already clinically available,safe, inexpensive and well-tolerated drug. Moreover, metformin has adifferent mechanism of action in activating AMPK than resveratrol andhence can have differential efficacy in modulation of downstreamtargets. Metformin is known to activate AMPK through multiple indirectmechanisms including LKB1 stimulation (Shaw et al., 2005) and inhibitionof AMP deaminase (Ouyang et al., 2011). In addition, in contrast toresveratrol which lacks bioavailability, metformin has a goodbioavailability and thus can be given orally which is a preferred routeof administration in humans. Hence, the present inventors investigatedif systemic application of metformin can prevent development ofincision-induced mechanical hypersensitivity and further hyperalgesicpriming following the plantar incision. Animals received a plantarincision on the left hindpaw. Metformin (30, 100, 150 or 200 μg) orvehicle was injected intra-peritonially for 4 days starting 2 days priorto the surgery. Mice with plantar incision that received vehicledisplayed long-lasting acute mechanical hypersensitivity (9 days) aswell as hyperalgesic priming following PGE₂ lasting at least 24 hrs. Incontrast, intra-peritonial injection of maximal dose (200 mg/kg) ofmetformin prevented both plantar-incision induced mechanicalhypersensitivity (FIGS. 2A and 2B) and the expression of hyperalgesicpriming following PGE₂ injection on day 14 (FIGS. 3A and 3B).Interestingly, the lower doses were not effective in blockingincision-induced acute mechanical hypersensitivity; however theysignificantly blocked hyperalgesic priming.

OSU53 Inhibits Acute Mechanical Hypersensitivity and HyperalgesicPriming in a Model of Post-Surgical Pain in a Dose-Related Manner.

Although metformin is a clinically available and safe drug, the exactmechanism of action of metformin in activating AMPK is still unknown.Moreover, the effects of metformin have been attributed toAMPK-independent mechanisms as well (Foretz et al., 2010). Recently, anovel AMPK activator with a distinct mechanism of action, OSU53, wassynthesized by scientists at Ohio State University (Lee et al., 2011).OSU53 stimulates AMPK kinase activity through direct activation withhigh potency (EC₅₀, 0.3 μM) independent of its upstream kinase LKB1 (Leeet al., 2011), a mechanism distinct from that of metformin (Fryer etal., 2002; Lee et al., 2011; Shaw et al., 2005). Moreover, OSU53 is apotent, orally bioavailable AMPK activator making it a potential drugfor treatment of post-surgical pain. The present inventors assessed ifOSU53 prevented the development of acute hypersensitivity andhyperalgesic priming following plantar incision. Animals received aplantar incision on the left hindpaw. OSU53 (10 or 30 μg) or vehicle wasinjected locally into the left hind-paw around the incision eitherimmediately following incision. Mice with plantar incision that receivedvehicle displayed long-lasting acute mechanical hypersensitivity as wellas hyperalgesic priming following PGE₂ lasting at least 24 hrs. Incontrast, intra-plantar injection of OSU53 dose-relatedly blocked bothplantar-incision induced initial mechanical hypersensitivity (FIGS. 4Aand 4B) and the expression of hyperalgesic priming after PGE₂ injectionon day 14 (FIGS. 5A and 5B).

A769662 Doesn't Inhibit Acute Mechanical Hypersensitivity and butInhibits Hyperalgesic Priming in a Model of Post-Surgical Pain:

Another direct activator of AMPK kinase is an investigational compound,A-769662. A-769662 activates AMPK both allosterically and inhibitingdephosphorylation of AMPK on Thr-172 (Sanders et al., 2007) and thisactivation is independent of upstream kinases (Goransson et al., 2007).The present inventors investigated if A-769662 prevented the developmentof acute hypersensitivity and hyperalgesic priming following plantarincision. Animals received a plantar incision on the left hindpaw.A-769662 (30 mg/kg) or vehicle was injected intra-peritonially for 4days starting 2 days prior to the surgery. Mice with plantar incisionthat received vehicle displayed long-lasting acute mechanicalhypersensitivity as well as hyperalgesic priming following PGE₂ lastingat least 24 hrs (FIGS. 6A and 6B). Interestingly, systemic A-769662 wasnot effective in blocking incision-induced acute mechanicalhypersensitivity (FIG. 6A); however it significantly blockedhyperalgesic priming following PGE₂ injection (FIG. 6B).

Co-Treatment with Systemic Metformin and Local Resveratrol InhibitsAcute Mechanical Hypersensitivity and Hyperalgesic Priming in a Model ofPost-Surgical Pain:

Although, all the AMPK activators individually entirely attenuate thedevelopment of the incision-induced hypersensitivity and hyperalgesicpriming at the respective maximal doses, an effective treatment strategywould be to utilize sub-efficacious doses of AMPK activators andinvestigate if the co-treatment is equally efficacious in attenuatingacute hypersensitivity and hyperalgesic priming following plantarincision. This strategy minimizes the unwanted side-effects of the drugswhich occur while utilizing the maximal doses. Hence, the presentinventors investigated if co-treatment of sub-efficacious dose ofmetformin and resveratrol can have added effect in attenuation ofincision-induced mechanical hypersensitivity and hyperalgesic primingcompared to their individual effects. Animals received a plantarincision on the left hind-paw and received treatment of vehicle,resveratrol or metformin alone or a co-treatment of metformin andresveratrol. Metformin (150 μg) was injected intra-peritonially for 4days starting 2 days prior to the surgery and resveratrol (3 μg) wasinjected in the left hind-paw around the incision on the day of thesurgery and 24 hrs post-surgery. Mice with plantar incision thatreceived vehicle displayed long-lasting acute mechanicalhypersensitivity as well as hyperalgesic priming following PGE₂. Micewhich received metformin or resveratrol alone displayed blunted acutehypersensitivity compared to vehicle treated mice but the effect was notsignificant (FIG. 7A). In contrast, co-treatment with systemic metforminand local resveratrol at individually sub-efficacious doses at the timeof incision significantly attenuated acute hypersensitivity (FIGS. 7Aand 7B) and hyperalgesic priming (FIGS. 8A and 8B) suggesting potentialsuper-additive effects of combined AMPK activator use.

AMPK Activators Don't Affect Wound Healing Negatively in a Model ofPost-Surgical Pain:

An important potential consideration in these experiments is that AMPKactivators would be expected to reduce protein translation and thereforemay negatively influence wound healing. This effect would have anegative impact on their clinical utility. To test if AMPK activatorsaffect the wound healing, we utilized either vehicle or maximal doses ofthe AMPK activators (resveratrol or metformin or OSU53) described in theprevious experiments and assessed wound healing with Hematoxylin & Eosin(H & E) staining (Lai et al., 2009). Skin samples were excised 3 and 7days after plantar incision to assess healing and wound closure,respectively. For the animals treated with resveratrol, no differenceswere noted in the wound size between the resveratrol and vehicle treatedgroups on Day 3 (FIG. 9A). By Day 7, the wound was completely closed inthe animals in both groups receiving vehicle or resveratrol (FIG. 9A).Similar results were obtained with intra-peritonial metformin orintra-plantar OSU53 treatment (FIGS. 9B and 9C). Thus, a lack of effecton with AMPK activators would suggest that these compounds would beunlikely to interfere with the wound healing process.

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As used herein, the term “about” refers to plus or minus 10% of thereferenced number.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference cited in the presentapplication is incorporated herein by reference in its entirety.

The foregoing discussion of the invention has been presented forpurposes of illustration and description. Although there has been shownand described the preferred embodiment of the present invention, it willbe readily apparent to those skilled in the art that modifications maybe made thereto which do not exceed the scope of the appended claims.Therefore, the scope of the invention is only to be limited by thefollowing claims. In some embodiments, the figures presented in thispatent application are drawn to scale, including the angles, ratios ofdimensions, etc. In some embodiments, the figures are representativeonly and the claims are not limited by the dimensions of the figures. Itis intended to obtain rights which include alternative embodiments tothe extent permitted, including alternate, interchangeable and/orequivalent structures, functions, ranges or steps to those claimed,whether or not such alternate, interchangeable and/or equivalentstructures, functions, ranges or steps are disclosed herein, and withoutintending to publicly dedicate any patentable subject matter. In someembodiments, descriptions of the inventions described herein using thephrase “comprising” includes embodiments that could be described as“consisting of”, and as such the written description requirement forclaiming one or more embodiments of the present invention using thephrase “consisting of” is met.

What is claimed:
 1. A method of reducing incision-induced hypersensitivity or incision-induced hyperalgesic priming in a subject, said method comprising administering to the subject a systemic dosage of metformin and a local dosage of resveratrol at or near a surgical incision site, wherein co-treatment with systemic metformin and local resveratrol reduces incision-induced hypersensitivity or incision-induced hyperalgesic priming, wherein the dosage of resveratrol is an individually sub-efficacious dose, and the dosage of metformin is an individually sub-efficacious dose.
 2. The method of claim 1, wherein the dosages of resveratrol and metformin are administered at a time that is between 24 hours before and 24 hours after the surgical incision is made.
 3. A method for inhibiting the development of chronic pain due to a surgical incision in a subject, said method comprising administering both a dosage of resveratrol and a dosage of metformin to the subject, wherein co-treatment of both resveratrol and metformin help inhibit the development of chronic pain from the surgical incision, wherein the dosage of resveratrol is an individually sub-efficacious dose, and the dosage of metformin is an individually sub-efficacious dose.
 4. The method of claim 3, wherein the doses of resveratrol and metformin are administered at a time that is between 24 hours before and 24 hours after the surgical incision is made.
 5. The method of claim 3, wherein resveratrol is administered locally to the incision and metformin is administered systemically. 